- Email: [email protected]
Lyme borreliosis
Journal of Clinical Microbiology and Infection, Volume 3 Supplement 2
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Conclusions: Complex studies on propemes of C.d. strains, circulating in Belarus, could be applied for the determination of pathogen origin, ways of its transmission, whch are an object of special importance for diphtheria surveillance.
I P593 I Detection and Differentiationof Leptospira sp.
Conclusions: Leptospirosis had a severe presentation in approximately 50% of the cases and a mortality rate of 20% of the global series. A high incidence of severe renal, pulmonary affectation, and rabdomiolysis was observed. The determination of IgM anti-leptospira by IgM-ELSA improves the efficiency of the usual serodganosis by IH, m a d y in the most serious forms ofleptospirosis.
by PCR Method M. Witkowska I , A. Samet I , T. Kocik ', A. Burluewicz ', J. Kur 3 . 'Dept. of Clinical Bacteriolofl, State Clinical Hospital W 1, Poland, Eterinary Hygiene Research Institute, Poland, 'Dept. of Microbiology, Echnical University of Gdansk, Poland
Objectives: Detection of Leptospira sp. in clinical specimens. Daerentiation of Leptospira serovars with (1)junction of two techniques: polymerase cham reaction (PCR) and restriction fkgment length polymorphm (RFLP), and (2) fingerprinting polymerase chain reaction (FP-PCR). Methods: Twenty seven cultures' of Lepfospira serovars and Leptospira icterohaemodagiae serially diluted strains (in blood, sperm and urine) were tested by PCR with Leptospira genus-specific primers. Experimentally infected blood samples were examined by the Same test. Nineteen Leptospira serovars were analyed by FP-PCR with L1 and G1 primers. The 16s rRNA of Leptospira s e r o m genes were amplified with PCR and analysed by RFLP with AfuI enzyme. The DNA was extracted by kits made by A&A Biotecbnology, Poland. Results: The Leptospira DNA was detected in urine, sperm and blood. The PCR test enables detection of 10 Leptospira cells in PCR probe and was genus-spec& for all tested samples. Total time of the test was less than 6 h. Leptospires can be detected in blood on second day of the dection. Both tests PCR-RFLP and FP-PCR gave partidly differentiation of Lqfospira semvars. Conclusion: Test based on PCR technique is useful for quickly, sensitive and specific detection of leptospires in blood, sperm, urine, and give very early diagnosis of leptospirosis. Both diflerentiating tests may be used in epidemiological studies as alternative, additional method.
I P594 I Early Serodiagnosis of Serious Forms of Leptospirosisby ISM-ELSA J.L. L6pez-Hontangas, M. Salavert, F. Ponz, I. CenicerosI , I. Gutierrez, M. Gobernado. Deparf?nentofMicrobiology and Intensive Care Unit, Hospital L4 FE, VaJencia, Spain
Objectives: To study the epidemiological and climcal findings of leptospirosis in our geographical area, and to evaluate the utility IgM-ELSA test to detect leptospire-specific immunoglobulinM for early serodragnosis of severe forms. Patients and Methods: Fifteen patients with clinical diagnosis of leptospirosis were evaluated. A technique of indirect hemagglutination (IH) (HilLrest B i d ) as test for screening was used. A title >1:50 was considered negative. To detect the IgM anti-leptospira specific of gender an ELISA (PanBio) was employed. Results: From January 1993 to December 1996 the diagnosis of leptospirosis was made in 15 patients out of 86 patients with initial suspicion. Seroconvenion or initial positive title of IH was detected in 8 patients. In 11 patients with initial negative or doubtfid titles of IH, IgM ELISA w a s positive in 9 of them. Of 9 patients with initial IH negative or doubtll and IgM-positive, 5 of they had serious dlsease. From all patients eight sdered a serious leptospirosis and three died. Four patients needed admission in a intensive care unit. During the clinical course renal failure was observed in 7 cases, rabdomiolyss in 6, tbrombopenia in 6, meningoencefalitis in 3 and shock with multiple organs failure in other 3.
Lyme borreliosis
1 P595 I Simultaneous Presence of Two Botrelia
burgdorfrrisensu lato Species in lxodes ricinus Ticks
J. Jenek. Knml Marcinkowski University ofMedical Sciencer in Poznari, Poland Hematophagous arthropods, lvodes ricinus ticks are transmitters of agent of Lyme disease Bonefia bufgdofm' to men and animals. Recently it has been demonstrated that DNA fiom more than one of the three Borrelia burgdorferi sensu lato species was present I. ricinus nymphs @chon et al., Emerging Infect. Dis., 1995; 1 (3): 89-90). We analyzed 15 h m 73 PCR positive for Bomlia DNA ticks genek and Glazaczow, Przeg. Epid., 1996; 50: 383-386) for simultaneous presence of more than one genospecies in unfed adult ticks by using PCR amplification of flagelin gene fiagment and hybridization with genospecies-specific probe. Thirteen ticks were dected by a smgle species of Bomlia (eigth by B. garinii and five by B. afzelii; Boneh burgdofm' sensu sfricfo was not found), and two were infected by both B. garinii and B. afzzdii. These p r e h a r y data suggest that the simultaneous presence of more than one genospecies in arthropod vector may be not exceptional.
lp5961 Botrelia burgdorfrm'sensulato DNA in Setum of Patients with Early Lyme Disease J. Jenek, A. Szkaradkiewicz, T. Tdecka. Karol Marcidmwski University ofMedical Sciences in PoznaA, Poland The studies aimed at evaluating dugnostic capacity of PCR technique and of serological techniques in erythema chronicum migrans (ECM) dlagnosis. Reaospective studies on Borrelia burgdot$+ (B.b.) DNA presence in serum of patients with early bomkosis were conducted using PCR and hybridzation with specific probe. F~fiyfour sera were tested earlier for presence of anti-B.b antibodies (Enzygnost Borreliosis, Behring). High titre of specific anti-B.b. antibodies were found in 9 sera (IgG antibodies in 3 sera, IgM antiborhes in 3 sera and in 3 sera both types of antibodies). In 7 cases specific activity of serum IgM was at the trace level. Presence of 8.6. DNA was noted in 16 sera of which 5 only contained specific anti-B.b. antibodies. The obtained results indicate that ECM diagnostics based on immunoserological techniques is insuflicient and in clinically suspected cases should be supplemented by PCR studies.
[ P597 I Lyme Disease as an EpidemiologicalProblem in Poland Z. Anusz,A. Horban, E. Marcinkowska, M. Poniewierski. I.fectious Diseaies Hospital, Warsaw, Poland The first serological study in group of a h g h risk was carried out in Poland in 1991 (Anusz et al.). In 61 out of 417 persons (14.6%) antibodies against Bomlia bufgdofm' were found.
Lyme borreliosis
Since 1996 borreliosis must be obligatory registered and reported to the Department of Public Health. In the first quarter 20 cases were reported, in the second 41 cases, in the third quarter 308 cases. One hundred seventeen patients (31.7%) were hospitalized. The biggest group of patients was reported in the forest areas: Bialystok 53 (14.4%),Olsztyn 30 (8.1%), Bydgoszcz 30 (8.1%), Suwalki 28 (7.6%). We presume that &SI number is underestimated. In 1995-1996 years in our hospital only we observed 188 patients with newly recognized Lyme disease. One hundred seventy six patients were addmmed with erythema migrans, eleven with symptoms of phase 11, one in phase 111.
F/ Lyme Disease among the lnhabitants of the North-Eastem Part of Poland S. Pancewicz, A. Januszkiewicz, E. Siwak, T. Hermanowska-Szpakowicz, J. Zajkowska, W. Krupa, M. Pryszmont. Department of Neuroinjction in Medical Academy in Bialystok, Poland The purpose of this study was to estimate fkquency of Borrelia burgdorferi antibodies detection among 1765 inhabitants in the north-eastern part of Poland. 1101 of whole number came fium a hgh risk group exposed to direct contact with the tick Ixodes ricinus (persons employed in the woods and lnhabitants of +es situated close to the woods). Active Lyme disease was diagnosed in 191 persons (45.0%).The rest 227 were antibody carriers (54.4%). Regarding the whole number of 1765 persons examined it equals adequately 10.8%of persons with active Lyme disease and 12.8% of antibody camers. Among 191 persons with systemic changes the following disorders were identified Erythema migrans (6.11%), Lyme arthritis in 87 persons (14.92%),Neuroborreliosis (1.92%), Lyme carditis in 11 (0.62%)keratitis in 1 (0.05%) and iritis in 1 (0.05%).
I P599 I Lyme Disease - One Year Follow-Up of 98 Pts E. Marcinkomka, A. Horban, Z. Anusz, H. Zarnowska. Hospitalfor Infectious Diseases, Warsay Poland We observed 98 persons (74 women, 23 men, range 15-80 y, mean range 47y). Ninety seven pts had erythema migrans. In 1 case fiat clinical symptoms suggested neuroboreliosis. Only 19 pts had other symptoms. Phase I1 of disease was observed in 9 cases: arthritis (7), neuroboreliosis (l), erythemas multiplices (l), phase 111 in 1 case. Antibodes were detected with ELISA (DAKOLYME) after 3, 6, 9 months of onset. Seventy seven patients were treated with Amoxicillin (1.54 g/d) for 21 days, 14 consecutive with Doxycycline (200 mg/d) for 21 days, 2 pts were treated by various regimens. Five p a were not treated in phase I. In phase I1 and 111 Doxycycline (200 mg/d) or Cefhinxone (2g/d) had been administered. Four pts needed retheram. None of seronegative pts had phase I1 and I11 symptoms, 9.2% of seropositive pts had phase I1 and 1%had phase 111. Among pts treated with Amoxicillin antibodies were found in 58% and phase I1 of dsease was observed in 5.1%. Among pts treated with Doxycycline there were found in 50% and 7.1% respectively. M e r 12 months of onset no one had any signs or symptoms of Lyme &ease. Conclusions: (1) Data suggest that an early antibiotic treatment prevents phase I1 of Lyme disease. (2) Both Amoxicillin and Doxycycline therapy seem to be good treatment in erythema migrans.
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1P600 I Immune Complexesin Course of Lyme Bomliosis S. Tylewska-Wierzbanowska, T. Chmielewski. Department hcteriolon, National Institute of Hygiene, Warsaw, Poland
of
Lyme borreliosis is a multi-step disorder with variety of symptoms and pathologic processes. It has been suggested that in course of chronic progressive Lyme borreliosis observed manifestations are a result of development of immunologically based disease and formation of immune complexes. Patients with c h c a l symptoms of Lyme borreliosis at dif€erent stages were tested. Level of serum antibodies specific to B. burgdofri was estunated with recombinant Igh4 and IgG ELISA (Biomedica, Austria) and blood samples were cultured to isolate spirochetes. In cases of negative results of serological testing, patients' sera were pre-treated to precipitate and dissociate immune complexes. After that treatment level of antibodies specific to B. burgdofen was evaluated. It has been found that in seronegative Lyme borreliosis patients antibodies to B. burgdofm' form immune complexes. The Lnfection has been confirmed by isolation of spirochetes h m blood. The detection of immune complexes formed by B. burgdofm' antibodies is a valuable test which can improve diagnosis of the disease and may be helpful in establishmg more complex treatment of the infection as well as it's sequelae.
I P601 I Evaluationof the Specific Cellular Immune Response to Bomlia burgdotii?ti Soluble Antigens in Lyme Disease
',
T. Malovrh', V. Kotnik A. Paternoster', E. Ruzic-Sabljic J. Cimperman E Sterle', A. K0tnik3. 'Institute ofMinobiology and
',
Immunolon, Medical Faculty Ljubljana, Slovenia, 'Department of Infectious Diseases, University Medical Center Ljubljana, Slovenia, 'Department .f Viroloa, Institute $Public Health ofthe Republic of Slovenia, Slovenia
Objectives: Detection of the infection with Borrelia burgdofri is still controversial. Clinical signs and the results of laboratory testings are not contributing enough to the diagnosis of the disease. In our laboratory we are performing for years specific lymphocjte blast transformation test (TTL) with Borrelia burgdo@i soluble antigens. The aim of the study was how this test contribute to the diagnosis of Lyme disease. Methods: We performed historical cohort study reviewing laboratory data (TTL) of 800 patients with clinical signs relevant for Lyme disease. 213 of them were were selected according to the clinical signs in five groups. The first group was composed of patients with verlhed erythema migrans, the second group of patients with cardial and neurological signs, the third group of patients with acrodermatitis chmnica atmphicans, arthritis and encephalomyelitis, in the fourth group were patients with non-specific, but with lyme disease often connected symptoms, and in the fifth group patients with signs belonging to more then one of the first four groups. Results: 45.8% of the patients in the first group had positive cellular immune response, 57.1% in the second group, 11.1% in the third group, and 22.1% in the fourth group. Patients fium the fifth group had positive cellular immune response in 37.2%. Conclusion: Available laboratory tests for cellular and humoral immunity can not give the straight forward answer characteristic for the efficient immune response to Bomlia burgdofm' mfection.
Journal of Clinical Microbiology and Infection, Volume 3 Supplement 2
142
[ E l Characterizationof Slovenian Lyme borreliosis
Erythema Migrans in the lmmunocompromised Host
spirochetes
E. Rukit-Sabljit I , E Strle’, J. Cimperman’, S. Lotrit’, V Maraspin2. ‘Inst. ofMicrobiology, Ljubljana, Slovenia, ’ D pt. Infectious Dis., Medical Faculty Ljubljana, Slovenia
Objectives: LB is the most prevalent tick-borne infection in Slovenia. The signs and symptoms of LB vary, the most common dinical manifestation is skin rash, EM. Disseminated infection includes severe neurologic, cadac, skin, and arthritic manifestations. Phenotypic characteristic and genetic Merendation of LB spirochete has been performed. In the present study, we analyzed-24 B. burgdorjm’ sensu lato s t r a i n s isolated from the skin of Slovenian patients. Methods: Skin biopsies were inoculated &to the MKP medium, incubated at 33°C and weekly examined for spirochetes by dark-field microscopy. Protein profdes were determined by PAGE after b o h g spirochetes with 2.5% SDS. Species identdication were determined by PFGE using Mlul restriction. Results: Mlul digestion profeiles showed that 20 (83.4%) isolates belong to B. afielii and 4 (16.6%) to B. garinii, none B. burgdorjeri sensu strict0 strain has been evidenced. All B. garinii strains have been isolated b m the patients with EM, while B. afielii strians were cultured: 15 b m EM, 3 f b m ACA and two f b m the s k i n of previous EM. Borreliae showed quantitative differences in the amount of expressed proteins, OspC was the most frequently hghly expressed. OspA and OspB proteins have been expressed by 19 (95%) B. a f i l i i and two (50%)B. garinii strains, OspC by 10 (50%)B. ofzelii and all B. garinii strians. Conclusions: In Slovenia, skin disorders are the most fiequent c h c a l madestations of LB and B. afielii is dominant strain from the LB spirochetes complex.
lP603( Comparison of Home-Made IFA with Commercial ELISA Assays for Diagnosing Lyme Disease A.M. Zore’ , E. RuZit-Sabljit E Sale’, J. Cimperman2, S. Lowit’, V Maraspin’. ‘Inst. ofMinobiology, Medial Faculty in Ljubljana, Slovenia, ’Dept. for Infectious diseases, Medicnl Faculty in Ljubljana, Slovenia Objectives: Cornparison of sensitivity and specificity of two commercially available ELISA tests and home-made IFA test for detecting IgG and IgM against Borrelia burgdo@ (BB). Methods: Serum samples of 198 patients suspected to have Lyme disease (LD) and of 214 healthy persons (HP) were tested with Lyme Behring ELISA (Behring, Mannheim, Germany), Dako Lyme Borreliosis ELISA (Dako,Gloshup, Denmark) and home-made IFA tests for IgG and IgM against BB. ELISA assays were performed according to the manufactures’ instructions. For IFA antigen w a s used s tr a i n of B. aftelii, the most fiequent Slovenian isolate. Results: The sera of group of healthy persons were IgM 97.2% negative by IFA, 82.7% negative by Behring ELISA and 90.6% by Dako ELISA. IgG were 87% negative by IFA, 56.3% negative by Behring ELISA and 84.9% negative by Dako ELISA. The sera of patients with erythema migrans were IgM 80% negative by IFA, 37.5% negative by Behring ELISA and 52.5% by Dako ELISA. IgG were in this group 80% negative by IFA, 57.5% negative by Behring ELISA and 65% by Dako ELISA The sera of patients with other manifestations of Lyme disease were IgM negative in 90% by IFA, 55.7%by Behring ELISA and 54.4% by Dako ELISA. IgG were 64.5% negative by IFA, 38.6% by Behring ELSA and 50% by Dako ELISA. Conclusions: The best sensitivity and specificity have been achieved by home-made IFA. The commercial ELISA tests should be used for serological screening because of its increased sensitivity.
V. Maraspin, E Strle, S. Lotrit-Furlan, J. Cimperman, E. Rukit-Sabljit. University Medical Centre, Ljubljana, Slovenia Objectives: To assess the course and the outcome of antibiotically treated immunocompromised patients (ICP) with erythema migrans (EM).
Methods: In the period b m 1991to 1995,49 adult patients with typical erythema migrans and previously identified immunocompromised condition were investigated and followed-up for one year at the Lyme borreliosis Outpatients’ Clinic, University Department of Infectious Diseases, Ljubljana, Slovenia. Results: There were 26 females and 23 males, aged from 17 to 77 years (medan 58 years), suffering from dfferent causes of immunodeficiency. The results were compared with 49 previously healthy age, sex and antibiotic therapy matched persons with an EM. Clinical characteristics of Lyme borreliosis (LB) before treatment were similar in both groups. The majority of patients were treated with an oral antibiotic, only three (6.1%) ICP and two (4.1%) controls received an i.v. antibiotic because of signs of early dissemination of LB. The median duration of EM after the institution of therapy was 10 days (2-90) in the ICP group and 14 d a y s (2-90) in the control group (nonsigdcant difference). Among the ICP five (10.2%) patients developed minor and three (6.1%) major manifestations of LB, while in the control group in three patients (6.1%) minor manifestations were observed and none developed major d e s t a t i o n s ; all required a retreatment with an i.v. antibiotic. Conclusions: The course and the outcome of early LB in immunocompromised patients seem to be sirmlar as in normal population.
[p605] CefuroximeAxetil (CEF), Doxycycline (DOX) and Amoxicillin (AM) for Treatment of Solitary Erythema Migrans (EM) E Strle, V. Maraspin, S. Lotrit-Furlan, E. Rukit-Sabljit, T. Jurca, J. Cimperman. University Medical Centre, Ljubljana, Slovenia Objectives: To compare effectiveness and adverse effects of cefuroxime axed, doxycydine and amoxicillin for weatment of EM. Methods: The study was prospective and began in 1994. For the presentation in this report 210 adult patients with previously untreated typical solitary EM were selected, i.e., the first 70 patients treated with CEF (500 mg bid for 15 d a y s ) , DOX (100 mg bid for 15 days), or AM (1000 mg tid for 5 days, followed by 500 mg tid for additional 10 days) who were followed-up for one year. Results: The median duration of s h lesions after the institution of treatment was 10 (3-21) days in the CEF group, 11 (3-30) days in the DOX group, and 9 (2-28) days in patients treated with AM (nonsigdcant diflerences). During the follow up of 12 months 2 patients (1 in DOX and 1 in AM group) developed major later manifestations of Lyme borrehosis, and in 42 (20%) patients minor manifestations appeared in 13 (18.6%) treated with CEF, in 17 (24.3%) receiving DOX, and in 12 (17.1%) given AM. Bomlia burgdorjm’ sensu lato w a s isolated from the periphery of EM lesions in 23/62, 30/65, and 33/68 patients before treatment with CEF, DOX, and AM, respectively. In 1/30 culture positive patient in DOX group, in 1/23 in CEF group and in 0/33 in AM group B. burgdorfoi sensu lato was isolated again b m normal appearing skin at the site of previous EM 2 months after institution of antibiotic therapy. Seven, 11, and 6 patients receiving CEF, D O X and AM, respectively, reported mild to moderate gastrointestinal discomfort. In addition, 8 patients ti-eated with DOX developed photosensitivity.
Lyme borreliosis
Conclusions: CEF, D O X and AM have similar effectiveness, but adverse effectsare sigtllficantlymore fkquent in patients treated with DOX.
I P606 I Treatment Failure in Erythema Migrans B. Sibanc, G. LegniLar, S. Cvitan,J. Blatnik. Department oflnfectious Diseases, General Hospital, Cefie, Slovenia
Objectives: We talk about erythema migrans (EM) treatment failure whenever we see: 1. EM persistence 2. EM reoccurrence, 3. persistence or development of extracutaneus sequelae (major signs and minor symptoms), 4. persistent increase of antibody titers, 5. persistence of Borrelia burgdoferi. Methods: EM has been treated in General hospital Celje - Infectious department since 1981. In years 1994-1996 544 lyme patients were treated. 323 of them had typical EM and consequent progress in lyme cksease, whlch inckcate the need for hospital treatment with ceftriakson (Lendacin). Among them, we found 51 properly treated EM patients, who show progress of illness. 28 patients were treated with azithromycin, 7 with doksicyclme, 6 with penicilin V, 4 with consequent administration of two proper antibiotics (ATB), 6 with other ATB-s. Results: Only 10 patients had minor signs at the time ofEM persistence and 13 were seronegative at the end of our study. 6 patients (5 children) developed disseminated EM, 11 arthritis, 3 meningoencephaiitis, 1 pericarditis, 1 facial palsy, 29 minor symptoms which persisted more than 3 months or even upgraded. Conclusions: AU patients were treated with cefniakson 2 g/d i.v. 2-4 weeks. After that time, 25 patients were cured, 20 patients improved partially but in 6 patients there were no signs of improvement
143
OspC wxs identified more often than OspA. In the late stage antibody response against OspA was more fiequent than against OspC. Among cross-reacted sera the greatest part of B. burgdoferi antigens were discerned h m syphilitic sera.
Marseilles Fever and Lyme Borreliosis L. Angelov, T. Rakadjieva, R. Komitova, M. Pishmisheva, I. Hristova. Higher Medical Institute, Plovdiv, Bulgaria Some of our recent epidemiological studies have shown that human cases of Lyme borreliosis (LB) are reported h m areas where the environmental conditions are not optimal for the life cycle of the tick species I. ricinus. These areas are recognised as foci of Marsedles fever. It is known that the symptoms of LB vary h m patient to patient and often mimic other conditions. For this reason it is &cult for the clinician to distinguish between the two infectious disease. Objective of the study: To analyze the clinical manifestations of LB and Marseilles fever in a random sample of patients. An effort is made to establish the best therapeutic approach to these patients. Methods: The techniques of indirect immunofluorescence and ELISA. Results: 22 patients living in an area recognised as endemic for Marseilles fever were studied. Some of them developed clinical symptoms of LB and were serologically positive for infection with B. burgdoferi. Some domestic animals also tested positive for LB and Marseilles fever. Conclusions: The epidemiological evidence presented in this study gives us the grounds to consider that coinfections with B. burgdo@ and R. conori are possible. A new approach to the treatment of such patients is outlined.
at all.
Disappearance of EM after treatment does not necessarily mean cure of Lyme borreliosis, even when we use "proper ATB-s" long enough
I P609 I Clinical Manifestationsof Lyme Disease in
I P607 I Electrophoreticand Immunologic
N. Trofimov V Scherba L. Titov , E. Danilov A.I. Korzan *. 'Byelomssian Research Institute for Epidemiology and M i ~ o b i o l o ~ , Minsk, Republic of Belarus, 'Clinical Hospital of Brest Region, Republic of Belarus
Belams
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Characterizationof6orrelia burgdohn Isolated in Bulgaria I. Christova, M. Polianova, L. Froloshka. National Center oflnfecfious Diseases, Sofa, Bulgaria Objectives: To basically characterize Bulgarian B. burgdoferi isolates as well as humoral immune response in Lyme dIsease patients against them. Methods: Thirteen Bulgarian B. burgdorferi isolates /11 h m I. rioinus ticks and 2 h m skin biopsies from Erythema migrans patients/ were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis /SDS-PAGE/ and by immunoblotting with sera h m patients with Lyme disease. Cross-reacted sera from patients with syphilis, leptospimsis and lupus erythematodes served to discriminate between specific and nonspecific bands in immunoblots. Results: Regarding Merent species-specific B. burgdoferi OspA/OspB electrophoretic migration, basic identification of the isolates was made. Early humoral immune response in Lyme disease patients was found to be directed mainly against OspC and 41 kDa antigen, and very rarely against OspA. In the late stage of the disease antibohes were proven especially against 41 kDa, 66 kDa, OspA and only in single cases against OspC. Conclusions: SDS-PAGE characterization of Bulgarian B. burgdorjki isolates revealed considerable predomination of B. garinii both among tick and skm isolates. By sera h m patients with Lyme disease the following major antigens in the isolates were proven 66 kDa, 41 kDa, OspA, OspC. In the early stage of the disease
',
Objectives: To study prehmnary data on characteristics and peculiarities of Lyme Disease (LD) course in Byelorussian population. Methods: 24 patients with LD were examined by us in 1995-1996. The disease was diagnosed on the basis of migrating erythema (ME) in anamnesis, revealed during a physician's examination or h m the patients complaints. It was verlfied by detecaon of antibodies against Borrelia burgdofm' in blood sera with the help of indirect immunofluorescence assay Results: ME was a most fiequently occurred symptom h m all clinical d e s t a t i o n s (87%). Cutaneous manifestations were accompanied by common infectious syndrome in 10 cases (47%), that was expressed in fever of different rates, as well as in head, muscle and joint pains. Disturbances in cardIo-vascular system were reported in 9 patients (38%), they were attended by unpleasant sensation and pains in heart. Various rates of blockade in conducting system and atrial extrasystoleswere also identified in elecaocardiogrammes.The signs of anicteric hepatitis in the form of nausea, feeling of heaviness, located in right hypochondrium, enlargement of liver size and elevation of transaminase levels 2-3 times were established in 3 patients (12%).Serous meningitis ofborrelia etiology was diagnosed in 4 patients (17%). Conclusions: ME and common infectious syndrome are main clinical signs of LD. Lower prevelance of other symptoms, which are typical at late stages, appears to be conditioned by the early administration of antibiotics to patients.
Journal of Clinical Microbiology and Infection, Volume 3 Supplement 2
144
1 P610 I Prevalence of Bomlia burgdohti Antibodies in Urban and High Risk Areas
D. Tuncer',D. Colak', E Saym2,D. O@inc', C. Ergin', B. Tuncer3, G. Mutlu'. 'Akdeniz University CIin. Microbiology D q t , Antalya, Turk% 'Ankara University Veterinary Faculty, Ankara, Turkey, 3~tare Hospital Finike, Turkey Objectives: An epidemiological study on Lyme disease was made by comparing the populations of mountain villages and city centers. Methods: 244 subjects h m mountainous areas and 124 subjects finm city centers were examined with respect to Lyme &ease. B. burgdofm' IgG antibodies were searched for in the sera of subjects by microelisa technique. Positive results were later confirmed by Western blot techmque. Ticks we^ collected h m goats and sheep, which are in close contact with the people in endemic regions, and presence of Ixodes species, which are the reservoir of B. burgdorfen' was inveswted. Results: By microelm, 22.1% of rural and 6.4% of urban populations were shown to have B. burgdorferi IgG antibodies. By Western blot 80.6%of positive cases confirmed to be positive. 80.4% of ticks were idenhfied as Ixodec ricinus Conclusion: This study suggests that Lyme disease might be considered as endemic in rural areas of Antalya.
I P611 I Standardization of Western Blotting in Lvme Borreliosis (LB) A. Lakos. Lyme Disease Center, Budapest, Hungary Objective: New standardization of Western bloning in LB, specification of specific and aspecific bands, using Borreha ahelii (BA) as antigen. Methods: The proteins of BA (ACA1) were separated by PAGE and blotted onto Immobilon membrane. The immune reaction was carried,outwith 100 serum samples each of the following clinically defined groups: erythema migrans - EM (l),Bannwarth-syndrome - BS (2), acrodermatitis chi. atrophicans - ACA (3), infants (4), healthy blood donors - HBD (5), forestry workers (6) and uveitis patients (7). Groups 1-3 represent Merent clinical manifestations of Lyme &ease, groups 4-7 served as control. IgM and IgG antibodies were tested separately. In group 2, CSF samples were also evaluated. Altogether 1600 tests were done. The intensitiy of bands was estimated and scored finom 0 to 3. Location of bands was defined with monoclonal antibodies. Statistical evaluation was done by EPINFO and SPSS PC. Results: IgM antibodies against both the 22 kD and 41 kD protein was found to be 100% spenfic. However, IgM testing has a low diagnostic value, because only five EM and nine BS patients had IgM but no IgG antibodies. Anti-flagellar (41 kD) and anti-39 kD IgG antibodies were not spec& at all, in contrast with many previous studies. Specfic bands were 22; 29; 35; 44; 47; 49; and 93 kD. The best specificity/sensitivity ratio was found when cut-off level was s e t up at score 1.5 (one strong plus one faint reaction at the above-mentioned bands). According to t h criteria, ~ ~ only three HBDs and no d a n t s were positive. The sensitivity of IgG tests among consecutive EM and BS patients was 40 and 63%. respectively. In BS, when both CSF and serum, IgG and IgM were evaluated, the sensitivity was 88%.After six weeks the first symptoms, all BS patients were positive as well Y the ACA patients. Conclusion: We found a specificity pattern difierent h m previous studies. Our results suggest that specific Western blot pattern may vary strain by s m i n and there is no universally valid specific Western blot bands. This work should be repeated for any borrelia isolate candidate for dugnostic purpose.
(p612(Ti-Borne Borreliosisor Infectious
Mononucleosis- A Diagnostic Challenge in Children
L. Krbkwi M. KapZkovi *, I. Stefflovi', J. Kunicki 9.G. Mendel Children's Hospital, Univmity of Bmo, Czech Republic, 'Hygiene Institute, Bmo, Czech Republic Objectives: To examine IgM seropositivity against Bonelia burgdorfm' (Bb) with nonspecific symptoms for tick-borne borreliosis in
pediatric patients. Methods: 217 children were seen h m 1994 to 1996 for IgM seropositivity against Bb. Erythema migram, borrelial lymphocyt o m , neuroborreliosis and Lyme arthritis were diagnosed in 165 children. We analysed 35 children for possibly cross-reacting antibodies to rheumatoid and antinuclear factor, to viral capsid and nuclear antigen for Epstein-Barr infection. Enzyme-linked immunosorbent assays were used to dqnose Bb or EB infection. Westernblots or IgM antibody capture tests are not used routinely in primary care practice. Results: 26 girls and 9 boys, ages 3 to 18 years with cervical lymphadenopathy, fever, fatigue, cephalea or tonsillitis were repeatedly examined. The atypical lymphocytes were found in 16/35 (46%)patients. 10/35 (29%) children showed elevated transaminases.20/33 (61%) children had positive IgM antibody to viral capsid antigen for infectious mononucleosis in the I. sample and only 6/35 (17%) had it in 11. During the convalescent phase 54%-83% children developed I g G antibodies to EB nuclear antigen. Antinuclear and rheumatoid factor were negative in all patients. Conclusions: Tick-borne borreliosis and EB infection may both be associated with lymphadenopathy or other common features of illness. Cases with nonspecific symptoms, IgM seropositivity to Bb and those with tick bite history lead to erroneous diagnosis of tick-borne borreliosis. IgM seropositivity to Bb in 35/217 (16%) children with infectious mononucleosis supports the knowledge about false-positive serology in diseases with polydonal B cell activation.
(p613j Evolution of the Immune Responsein M i e n t s with Erythema Migrans G. Grabher, B. Redl, P.Loidl, G. Sto5er. Instifute o f M i m b i o l o ~ , Uniwrsity oflnnsbruck, Medical S h l , Austria Objectives: To assess the evolution of the immune-responses of patients with EM during the course of disease using immunoblots (IB) with extracts from three Borrelia genotypes and recombinant proteins. Methods: A total of 259 serially collected sera b m 97 phyucian documented patients with known duration of EM were analyzed by immunoblotcing for IgM and IgG, using extracts b m strains B31 (B. burgdofm' sensu stricto), PKo (B. afzeliil), PBi (B. garinit), recombinant proteins OspA, OspB, OspC, flagellin (and two Merent central hgments P41i), P39 and P100/83 h o r n B. garinii and a commercial DotBlot. Evaluation was, if applicable, as recommended by CDC and ASTPHLD. Results: From 45 patients which presented with EM of less than 7 days in duration 8 (18%) were IgM positive in IB,whereas h r n 52 patients with EM of 7-14 days in duration 47 (90%) were IgM positive. In total, the serum samples b m 88 (91%) of the patients revealed a seroconversion within 2-4 weeks after appearance of EM. IgM to OspC and flagellin remained detectable for long periods, up to 6 month in some patients. Conclusions: The appearance of IgM in Il3 correlated directly with duration of EM. The Merences in the reactivities with OspC
Lyme borreliosis
in dependence of the genospecies used for IB were generally compensated by detection of antibodies to P39, P41i and/or P100/83.
I P6141
Urinary Excretion of 6.burgdorfefiDNA in Patients with Lyme Borreliosis
B.L. Schmidt, E Breier, E. Aberer. L B I f dermato-wenerolog, Serodiagnosis, Vienna, Ausfria Objectives: To determine the urinary excretion of B. burgdoferi (Bb)-DNA in patients with Lyme borreliosis after therapy. Methods: Urine samples (n = 442) h m 134 patients (pat.) with uncomplicated erythema migrans (EM) or with EM accompanied by systemic symptoms, fever, chills, paresthesia, arthralgia or myalgia @MA), were tested before and up to one year after therapy by a nested PCR for the presence of Bb-specific DNA. Results: Immediately after end of therapy, 79% of pat. were positive by PCR. After 6 months, all but 9 were non reactive. Reevaluation of these 9 pat. showed interruption of treatment or treatment duration of only 5-10 days in 7 pat.. All could be retreated for 20 days with ceftriaxon and urine controls after three months were negative by PCR. Conclusion: Sensitive PCR-tests can demonstrate Bb-DNA in urine of pat. after matment. A persistent reactivity six months later indicates treatment failures.
I P615 I
Systemic Reaction in Patients Treated with Cefotaxime for Lyme Disease
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I P616 I Detection of 60rrelia burgdohriDNA in Muscle of Patients with Chronic Myalgias Related to Lyme Disease
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M. Frey I.', J. Sibha 3 , Y. PiCmont L. Marcellin ', E? Boohs , E? Vautravers M. Jesel', J.L. Kuntz H. Montes', B. Jaulhac '.
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'Services de Mbdecine Physique et Rhadaptation, Strarboutg, France, 'd'Explorations Fom'onnelles Neuromusculaires, Strarbourg, France, 'de Rhumatologie, Strasbourg, France, 4d'Anatomo-Pathologie Ghnhafe, Strarboutg, France, 'Institut de Bactk.iologie, Strarbourg, France
Objectives: Chronic myalgias related to Lyme disease are generally considered as sequelae. Our objectives were to establish whether Bonelia burgdorferi (E?. burgdoferi) may persist in the muscle of patients in such a case and to evaluate the contribution of a PCR assay for detecting this bacteria. Methods: In a prospective case-control study, B . burgdoferi DNA detection was performed by PCR on muscle biopsies of 8 consecutive patients with chronic widespread myalgias related to Lyme disease. The PCR assay used a specific chromosomal fragment of the flagellin gene. Other classical causes of myalgias were ruled out. Muscle samples from 14 controls were also tested. Results: B . burgdo& DNA was detected by PCR in muscle fium 4 of the 8 patients but not in controls. Three of the 4 positive patients had previously received an antibiotic treatment for Lyme disease; PCR analysis was done 3 months after antibiotic treatment for 2 of these 3 patients and 38 months for the thud one. Conclusion: Chronic myalgm related to Lyme disease can be associated with persistence of B. burgdofm' DNA in muscle, even despite previous supposedly curative antibiotic treatment.
Ch. Jvlichel J.-E? Collet2. ' D u g Surveillance Dept., Hoe& AG, Frankfurt, Germany, 'Dept. of Epidemiology and Biostatistirs McGill University, Montreal, Canada
m]
Objectives: Starting in 1989 a couple of adverse reaction reports were received fium the US in which the occurrence of a new systemic reaction - also reported as "delayed Jarisch-Herxheimer Reaction" - was idenhfied in patients treated with cefotaxime for Lyme disease. This study was undertaken to investigate whether this reported systemic reaction could be considered as a new adverse drug reaction to cefotaxime or the expression to the underlying Lyme disease. Methods: All spontaneous adverse reactions reports received by Hoechst AG h m the US within the period 01/Jan/87 to 31/Dec/93 were reviewed. An operational definition of the observed systemic reaction was established and reports were classified in five categories according to the probabihty to represent a case of the reaction: certain, possible, unlikely, highly unlikely or excluded. Results: A strong association between Lyme &ease as the indication for cefotaxime treatment and the systemic reaction was detected 97% (30/31) of the cases identified as certain were treated for Lyme disease, as opposed to 90% (27/30), 69% (31/45), 39% (651167) and 11% (42/392) of those idenhfied, respectively, as possible, unlikely, highly unlikely and excluded. Conclusions: This suggests that the new reported systemic adverse event may not be due to cefotaxime on its own but that it may either represent a combined effect of the drug and disease or persistent symptoms of the underlying Lyme disease not resolved by antibiotic theram. The possibility of an effect of long treatment duration of cefotaxime cannot be completely ruled out, but remains very unlikely.
I. Nahimana , 0. Peter *, L. Gern G. Praz F? Francioli . 'CHVY Lausanne, Switzerland, 'Insf. Central des Hbpitaur bblaisans, Sion, Switzerland, Unioersid de NeucMtel, Switzerland
Risk of Lyme Bomliosis after a Tick Bite in the French Part of Switzerland
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Objectives: To evaluate the risk of acquiring Lyme borreliosis after a tick bite and to correlate the risk with the presence of Borrelia sp
in the ticks. Method Since 1993, physicians of the French part of Switzerland were asked to report on all patients consulting for a tick bite. The protocol included a standardized questionnaire, two blood samples collected two months apart and mailing of the tick if available. Serology was performed with both a commercial test (Lyme IgG + M, Vidas Biomerieux) and a westernblot assay detecting both IgG and IgM. Criteria for positive immunoblot were a minimum of 5 bands including flagelhe and 2 of the following specific bands: P39, OspA, OspC 93 kDa. Seroconvenionwas defined by passing h m a negative immunoblot result to a positive one at the second sampling. Ticks were analyzed either by immunofluorescence or PCR with species-specific primers. Results: Among 510 patients who had a negative serology at the first visit, 40 (8%) seroconverted. The rate of seroconversion was 4% in the 297 patients who consulted because of the tick bite only (group I), 7.5% in the 121 patients with a s m a l l local reaction at the site ofthe bite (group 2) and 21% in the 92 patients who already had a lesion qu-g (25 cm) for an erythema migrans (EM): (group 3). Among the 418 patients of group 1 and 2, 8 (2%) developed an EM, among which 4 seroconverted. Among 179 analyzed ticks, 26 were positive. No correlation could be found between Bonelia sp in ticks, and clinical and/or serological results. Conclusions: In patients consulting for a tick bite without EM (group 1 and 2), the rates of seroconversion and EM were 5% and 2%, respectively. Seroconvenion and subsequent EM was more ke-
Journal of Clinical Microbiology and Infection, Volume 3 Supplement 2
146
quent in patients with small local lesions (group 2). suggesting that these lesions often represent Bonefia sp infection at an early stage.
I
Conclusion: The protein recognized by D6-Mab is Uely an outer surface protein (OSP) so far undescribed and we are in the process of characterizing it, knowing its imporrance for species typing and dagnosis.
[P618 Association of Distinct Clinical Manifestationsin Chronic Lyme Bomliosis with Bonelia burgdorhri sensu stricto, B. garinii and B. afzelii as Evaluated by lmmunoblot
0. Peter, A.G. Brea, E. Dayer. Institut Central des HGpitaux Ihlaisans, Sion, Switzerland
Objective: To evaluate the serologic reactivity ofpatients with Lyme arthritis, neuroborreliosis and Acrodermatitis chronica atrophicans (ACA) by immunoblots using B. burgdofm' sensu strict0 (ss), B. garinii and B. afrelii as antigens. Methods These 3 borrelia species are transmitted by Ixodes ricinus in Europe. A total of 81 patients with chronic symptoms of Lyme borreliosis (28 patients with arthritis, 28 with neuroborreliosis, 25 with ACA) were analysed by immunoblots using B. burgdofen ss, B . garinii and B. afzelii as antigens. In order to give a semi-quantitative appreciation of the reactivity, we scored (0 to 3 points) the intensity of the reaction to seven borrelia antigens ranging from 18 kDa to 93 kDa. Results: The serologic reactivity of patients with chronic manifestations of Lyme borreliosis was as follow: 62% of patients with neuroborreliosis reacted more intensively with B. garinii, 71% of patients with arthritis reacted with B . burgdogm' ss and 80% of patients with ACA reacted with B. afzelii. Usually criteria for positive immunoblot were reached whatever the Borreha species was. The stronger reactivity for one species was visually obvious and scores helped to objectivate the readings. Conclusions: These associations between the symptoms and the preferential serologic reactions to Werent Borrelia species may give additional arguments for the diagnosis of chronic Lyme borreliosis.
I P619 I Characterization of a Low Molecular Weight Lipoprotein Specific of B. garinii, Recognized by the D6-Mab E. Dayer, A.G. Brea, 0. Peter. Imtitut Central des HGpitaux Valaisans, Sion, Switzerland
Objective: To characterize the epitope recognized by D6-Mab specific of B. garinii. Introduction and Methods: Specific symptoms of chronic Borrekosis have been related to persistent infection with Herent Borrelia species. In Europe, neuroborreliosis has often been associated with B . garinii infections, while other symptoms, acrodermatitis chronica amphians (ACA) and arthritis, have been associated with B. afrelii and B. burgdofm', respectively. Typing the Werent isolates by their reactivity to spec& monoclonal antibodies is possible by immunoblotting. We used standard SDS-PAGE 1D and 2D gels with PVDF membrane transfer, as well as N-terminal sequencing to characterize protein. Results: D6-Mab recognized specifically B. garinii; the specific antigen is a surface lipoprotein of 11.2 kD and an estimated pkl of 5.7. Its N-terminal portion sequenced h m PVDF transfer membrane gave the following sequence: MVKKEAIIKAVNGLLVXP: The search for homologies revealed no identity and homologies of only 40% with POL-SOW3 non structural protein and with other ribosomal and peripksmic proteins. Using polyclonal antisera from chronically infected patients, we were able to show a major reactive protein in the same kD range in other Borrelia species, although it migrates in slightly Nerent position according to species.
1 P620 1 Developmentand Evaluationof Confirmatory Tests for Lyme Disease Using an Extract of B. garinii
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K. Gimrdet L. Binet', 0.Peter'. 'Imtifut Central des Hdpitaux Vafaisans,Sion, Switzerland, 'Lubodia S.A., Em, Switzerland Lyme hsease serology remains extcemely complex and positive resdts by ELISA require confirmatory tests (immunoblot or PCR). Most commercial confirmatory tests currently available are h m US origin and use as target antigens e m c t s from Borrelia burgdorferi. In Western Europe, s t r a i n prevalence is Herent and w e have developed two immunoblots (IgG and IgM) with an extract of B . garinii. These kin allow the identification of antibodies against proteins of 93, 60, 41, 39, 37,32.5,22-23 and 18 kD. C h c a l tr& organized in Switzerland have shown large geographcal variations in the seropositivity rate of the overall population. The speciticity of the luts has been evaluated for possible cross-reactions with syphilis or EBV antibodies and with autoantibodies. The sensitivity of the IgG immunoblot reaches 100% for Lyme arthritis, 96% for acrodermatitis, 90% for erythema migrans and 89% for neuroborreliosis.
Toxoplasma gondii
I P621 I
Prevalence of Toxoplasma Gondii Antibodies in Young Females of La Plata, Argentina
J. C o r d h i , M. Rub, G. Alberich, J. Contarelli, L. Massera, M. Michaan, M. Garcia, S. Cabrera. Infecous Diseases Department, Hospital San Juan de Dios, La Plata,Argentina Objective: To determine the seroprevalence of Toxoplasma gondii (TG) antibodies in urban and suburban young females finom La Plata, Argentina. Subjects and Methods: Samples from 271 consecutive healthy females were collected between February to October 1995. Serum was tested with indirect Hemaglutination with and without 2-mercaptoethanol. Positive test were confirmed with enzyme linked immunoassay. Results: The meda age was 22 (range 15-45). 156 (57.56%)living in the city, and 115 (42.43%) in a rural area close to La Plata. No Werences were found for social, cultural, religious or economical conditions between the groups. Dietary habits and frequency of contact with atmnals (including pets) were Werent (p 0.005). A global seroprevalence of 43.5% (118/271) was found. Values were higher in the rural group: 53.5% than in the urban (36.8%) p > 0.009. Moreover, the prevalence increase with the age, with significative a e r e n c e up to 33 yrs. (p = 0.008). Commentary: Suburban residence enhance the possibility to get TG as simJar as dietary habits and close contact with animals. The seroprevalence raise with the age. Because 56.5% of females were noreactives at wedding time, screening programs are necessary for pre-nuptial detection.
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Conclusions: Complex studies on propemes of C.d. strains, circulating in Belarus, could be applied for the determination of pathogen origin, ways of its transmission, whch are an object of special importance for diphtheria surveillance.
I P593 I Detection and Differentiationof Leptospira sp.
Conclusions: Leptospirosis had a severe presentation in approximately 50% of the cases and a mortality rate of 20% of the global series. A high incidence of severe renal, pulmonary affectation, and rabdomiolysis was observed. The determination of IgM anti-leptospira by IgM-ELSA improves the efficiency of the usual serodganosis by IH, m a d y in the most serious forms ofleptospirosis.
by PCR Method M. Witkowska I , A. Samet I , T. Kocik ', A. Burluewicz ', J. Kur 3 . 'Dept. of Clinical Bacteriolofl, State Clinical Hospital W 1, Poland, Eterinary Hygiene Research Institute, Poland, 'Dept. of Microbiology, Echnical University of Gdansk, Poland
Objectives: Detection of Leptospira sp. in clinical specimens. Daerentiation of Leptospira serovars with (1)junction of two techniques: polymerase cham reaction (PCR) and restriction fkgment length polymorphm (RFLP), and (2) fingerprinting polymerase chain reaction (FP-PCR). Methods: Twenty seven cultures' of Lepfospira serovars and Leptospira icterohaemodagiae serially diluted strains (in blood, sperm and urine) were tested by PCR with Leptospira genus-specific primers. Experimentally infected blood samples were examined by the Same test. Nineteen Leptospira serovars were analyed by FP-PCR with L1 and G1 primers. The 16s rRNA of Leptospira s e r o m genes were amplified with PCR and analysed by RFLP with AfuI enzyme. The DNA was extracted by kits made by A&A Biotecbnology, Poland. Results: The Leptospira DNA was detected in urine, sperm and blood. The PCR test enables detection of 10 Leptospira cells in PCR probe and was genus-spec& for all tested samples. Total time of the test was less than 6 h. Leptospires can be detected in blood on second day of the dection. Both tests PCR-RFLP and FP-PCR gave partidly differentiation of Lqfospira semvars. Conclusion: Test based on PCR technique is useful for quickly, sensitive and specific detection of leptospires in blood, sperm, urine, and give very early diagnosis of leptospirosis. Both diflerentiating tests may be used in epidemiological studies as alternative, additional method.
I P594 I Early Serodiagnosis of Serious Forms of Leptospirosisby ISM-ELSA J.L. L6pez-Hontangas, M. Salavert, F. Ponz, I. CenicerosI , I. Gutierrez, M. Gobernado. Deparf?nentofMicrobiology and Intensive Care Unit, Hospital L4 FE, VaJencia, Spain
Objectives: To study the epidemiological and climcal findings of leptospirosis in our geographical area, and to evaluate the utility IgM-ELSA test to detect leptospire-specific immunoglobulinM for early serodragnosis of severe forms. Patients and Methods: Fifteen patients with clinical diagnosis of leptospirosis were evaluated. A technique of indirect hemagglutination (IH) (HilLrest B i d ) as test for screening was used. A title >1:50 was considered negative. To detect the IgM anti-leptospira specific of gender an ELISA (PanBio) was employed. Results: From January 1993 to December 1996 the diagnosis of leptospirosis was made in 15 patients out of 86 patients with initial suspicion. Seroconvenion or initial positive title of IH was detected in 8 patients. In 11 patients with initial negative or doubtfid titles of IH, IgM ELISA w a s positive in 9 of them. Of 9 patients with initial IH negative or doubtll and IgM-positive, 5 of they had serious dlsease. From all patients eight sdered a serious leptospirosis and three died. Four patients needed admission in a intensive care unit. During the clinical course renal failure was observed in 7 cases, rabdomiolyss in 6, tbrombopenia in 6, meningoencefalitis in 3 and shock with multiple organs failure in other 3.
Lyme borreliosis
1 P595 I Simultaneous Presence of Two Botrelia
burgdorfrrisensu lato Species in lxodes ricinus Ticks
J. Jenek. Knml Marcinkowski University ofMedical Sciencer in Poznari, Poland Hematophagous arthropods, lvodes ricinus ticks are transmitters of agent of Lyme disease Bonefia bufgdofm' to men and animals. Recently it has been demonstrated that DNA fiom more than one of the three Borrelia burgdorferi sensu lato species was present I. ricinus nymphs @chon et al., Emerging Infect. Dis., 1995; 1 (3): 89-90). We analyzed 15 h m 73 PCR positive for Bomlia DNA ticks genek and Glazaczow, Przeg. Epid., 1996; 50: 383-386) for simultaneous presence of more than one genospecies in unfed adult ticks by using PCR amplification of flagelin gene fiagment and hybridization with genospecies-specific probe. Thirteen ticks were dected by a smgle species of Bomlia (eigth by B. garinii and five by B. afzelii; Boneh burgdofm' sensu sfricfo was not found), and two were infected by both B. garinii and B. afzzdii. These p r e h a r y data suggest that the simultaneous presence of more than one genospecies in arthropod vector may be not exceptional.
lp5961 Botrelia burgdorfrm'sensulato DNA in Setum of Patients with Early Lyme Disease J. Jenek, A. Szkaradkiewicz, T. Tdecka. Karol Marcidmwski University ofMedical Sciences in PoznaA, Poland The studies aimed at evaluating dugnostic capacity of PCR technique and of serological techniques in erythema chronicum migrans (ECM) dlagnosis. Reaospective studies on Borrelia burgdot$+ (B.b.) DNA presence in serum of patients with early bomkosis were conducted using PCR and hybridzation with specific probe. F~fiyfour sera were tested earlier for presence of anti-B.b antibodies (Enzygnost Borreliosis, Behring). High titre of specific anti-B.b. antibodies were found in 9 sera (IgG antibodies in 3 sera, IgM antiborhes in 3 sera and in 3 sera both types of antibodies). In 7 cases specific activity of serum IgM was at the trace level. Presence of 8.6. DNA was noted in 16 sera of which 5 only contained specific anti-B.b. antibodies. The obtained results indicate that ECM diagnostics based on immunoserological techniques is insuflicient and in clinically suspected cases should be supplemented by PCR studies.
[ P597 I Lyme Disease as an EpidemiologicalProblem in Poland Z. Anusz,A. Horban, E. Marcinkowska, M. Poniewierski. I.fectious Diseaies Hospital, Warsaw, Poland The first serological study in group of a h g h risk was carried out in Poland in 1991 (Anusz et al.). In 61 out of 417 persons (14.6%) antibodies against Bomlia bufgdofm' were found.
Lyme borreliosis
Since 1996 borreliosis must be obligatory registered and reported to the Department of Public Health. In the first quarter 20 cases were reported, in the second 41 cases, in the third quarter 308 cases. One hundred seventeen patients (31.7%) were hospitalized. The biggest group of patients was reported in the forest areas: Bialystok 53 (14.4%),Olsztyn 30 (8.1%), Bydgoszcz 30 (8.1%), Suwalki 28 (7.6%). We presume that &SI number is underestimated. In 1995-1996 years in our hospital only we observed 188 patients with newly recognized Lyme disease. One hundred seventy six patients were addmmed with erythema migrans, eleven with symptoms of phase 11, one in phase 111.
F/ Lyme Disease among the lnhabitants of the North-Eastem Part of Poland S. Pancewicz, A. Januszkiewicz, E. Siwak, T. Hermanowska-Szpakowicz, J. Zajkowska, W. Krupa, M. Pryszmont. Department of Neuroinjction in Medical Academy in Bialystok, Poland The purpose of this study was to estimate fkquency of Borrelia burgdorferi antibodies detection among 1765 inhabitants in the north-eastern part of Poland. 1101 of whole number came fium a hgh risk group exposed to direct contact with the tick Ixodes ricinus (persons employed in the woods and lnhabitants of +es situated close to the woods). Active Lyme disease was diagnosed in 191 persons (45.0%).The rest 227 were antibody carriers (54.4%). Regarding the whole number of 1765 persons examined it equals adequately 10.8%of persons with active Lyme disease and 12.8% of antibody camers. Among 191 persons with systemic changes the following disorders were identified Erythema migrans (6.11%), Lyme arthritis in 87 persons (14.92%),Neuroborreliosis (1.92%), Lyme carditis in 11 (0.62%)keratitis in 1 (0.05%) and iritis in 1 (0.05%).
I P599 I Lyme Disease - One Year Follow-Up of 98 Pts E. Marcinkomka, A. Horban, Z. Anusz, H. Zarnowska. Hospitalfor Infectious Diseases, Warsay Poland We observed 98 persons (74 women, 23 men, range 15-80 y, mean range 47y). Ninety seven pts had erythema migrans. In 1 case fiat clinical symptoms suggested neuroboreliosis. Only 19 pts had other symptoms. Phase I1 of disease was observed in 9 cases: arthritis (7), neuroboreliosis (l), erythemas multiplices (l), phase 111 in 1 case. Antibodes were detected with ELISA (DAKOLYME) after 3, 6, 9 months of onset. Seventy seven patients were treated with Amoxicillin (1.54 g/d) for 21 days, 14 consecutive with Doxycycline (200 mg/d) for 21 days, 2 pts were treated by various regimens. Five p a were not treated in phase I. In phase I1 and 111 Doxycycline (200 mg/d) or Cefhinxone (2g/d) had been administered. Four pts needed retheram. None of seronegative pts had phase I1 and I11 symptoms, 9.2% of seropositive pts had phase I1 and 1%had phase 111. Among pts treated with Amoxicillin antibodies were found in 58% and phase I1 of dsease was observed in 5.1%. Among pts treated with Doxycycline there were found in 50% and 7.1% respectively. M e r 12 months of onset no one had any signs or symptoms of Lyme &ease. Conclusions: (1) Data suggest that an early antibiotic treatment prevents phase I1 of Lyme disease. (2) Both Amoxicillin and Doxycycline therapy seem to be good treatment in erythema migrans.
141
1P600 I Immune Complexesin Course of Lyme Bomliosis S. Tylewska-Wierzbanowska, T. Chmielewski. Department hcteriolon, National Institute of Hygiene, Warsaw, Poland
of
Lyme borreliosis is a multi-step disorder with variety of symptoms and pathologic processes. It has been suggested that in course of chronic progressive Lyme borreliosis observed manifestations are a result of development of immunologically based disease and formation of immune complexes. Patients with c h c a l symptoms of Lyme borreliosis at dif€erent stages were tested. Level of serum antibodies specific to B. burgdofri was estunated with recombinant Igh4 and IgG ELISA (Biomedica, Austria) and blood samples were cultured to isolate spirochetes. In cases of negative results of serological testing, patients' sera were pre-treated to precipitate and dissociate immune complexes. After that treatment level of antibodies specific to B. burgdofen was evaluated. It has been found that in seronegative Lyme borreliosis patients antibodies to B. burgdofm' form immune complexes. The Lnfection has been confirmed by isolation of spirochetes h m blood. The detection of immune complexes formed by B. burgdofm' antibodies is a valuable test which can improve diagnosis of the disease and may be helpful in establishmg more complex treatment of the infection as well as it's sequelae.
I P601 I Evaluationof the Specific Cellular Immune Response to Bomlia burgdotii?ti Soluble Antigens in Lyme Disease
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T. Malovrh', V. Kotnik A. Paternoster', E. Ruzic-Sabljic J. Cimperman E Sterle', A. K0tnik3. 'Institute ofMinobiology and
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Immunolon, Medical Faculty Ljubljana, Slovenia, 'Department of Infectious Diseases, University Medical Center Ljubljana, Slovenia, 'Department .f Viroloa, Institute $Public Health ofthe Republic of Slovenia, Slovenia
Objectives: Detection of the infection with Borrelia burgdofri is still controversial. Clinical signs and the results of laboratory testings are not contributing enough to the diagnosis of the disease. In our laboratory we are performing for years specific lymphocjte blast transformation test (TTL) with Borrelia burgdo@i soluble antigens. The aim of the study was how this test contribute to the diagnosis of Lyme disease. Methods: We performed historical cohort study reviewing laboratory data (TTL) of 800 patients with clinical signs relevant for Lyme disease. 213 of them were were selected according to the clinical signs in five groups. The first group was composed of patients with verlhed erythema migrans, the second group of patients with cardial and neurological signs, the third group of patients with acrodermatitis chmnica atmphicans, arthritis and encephalomyelitis, in the fourth group were patients with non-specific, but with lyme disease often connected symptoms, and in the fifth group patients with signs belonging to more then one of the first four groups. Results: 45.8% of the patients in the first group had positive cellular immune response, 57.1% in the second group, 11.1% in the third group, and 22.1% in the fourth group. Patients fium the fifth group had positive cellular immune response in 37.2%. Conclusion: Available laboratory tests for cellular and humoral immunity can not give the straight forward answer characteristic for the efficient immune response to Bomlia burgdofm' mfection.
Journal of Clinical Microbiology and Infection, Volume 3 Supplement 2
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[ E l Characterizationof Slovenian Lyme borreliosis
Erythema Migrans in the lmmunocompromised Host
spirochetes
E. Rukit-Sabljit I , E Strle’, J. Cimperman’, S. Lotrit’, V Maraspin2. ‘Inst. ofMicrobiology, Ljubljana, Slovenia, ’ D pt. Infectious Dis., Medical Faculty Ljubljana, Slovenia
Objectives: LB is the most prevalent tick-borne infection in Slovenia. The signs and symptoms of LB vary, the most common dinical manifestation is skin rash, EM. Disseminated infection includes severe neurologic, cadac, skin, and arthritic manifestations. Phenotypic characteristic and genetic Merendation of LB spirochete has been performed. In the present study, we analyzed-24 B. burgdorjm’ sensu lato s t r a i n s isolated from the skin of Slovenian patients. Methods: Skin biopsies were inoculated &to the MKP medium, incubated at 33°C and weekly examined for spirochetes by dark-field microscopy. Protein profdes were determined by PAGE after b o h g spirochetes with 2.5% SDS. Species identdication were determined by PFGE using Mlul restriction. Results: Mlul digestion profeiles showed that 20 (83.4%) isolates belong to B. afielii and 4 (16.6%) to B. garinii, none B. burgdorjeri sensu strict0 strain has been evidenced. All B. garinii strains have been isolated b m the patients with EM, while B. afielii strians were cultured: 15 b m EM, 3 f b m ACA and two f b m the s k i n of previous EM. Borreliae showed quantitative differences in the amount of expressed proteins, OspC was the most frequently hghly expressed. OspA and OspB proteins have been expressed by 19 (95%) B. a f i l i i and two (50%)B. garinii strains, OspC by 10 (50%)B. ofzelii and all B. garinii strians. Conclusions: In Slovenia, skin disorders are the most fiequent c h c a l madestations of LB and B. afielii is dominant strain from the LB spirochetes complex.
lP603( Comparison of Home-Made IFA with Commercial ELISA Assays for Diagnosing Lyme Disease A.M. Zore’ , E. RuZit-Sabljit E Sale’, J. Cimperman2, S. Lowit’, V Maraspin’. ‘Inst. ofMinobiology, Medial Faculty in Ljubljana, Slovenia, ’Dept. for Infectious diseases, Medicnl Faculty in Ljubljana, Slovenia Objectives: Cornparison of sensitivity and specificity of two commercially available ELISA tests and home-made IFA test for detecting IgG and IgM against Borrelia burgdo@ (BB). Methods: Serum samples of 198 patients suspected to have Lyme disease (LD) and of 214 healthy persons (HP) were tested with Lyme Behring ELISA (Behring, Mannheim, Germany), Dako Lyme Borreliosis ELISA (Dako,Gloshup, Denmark) and home-made IFA tests for IgG and IgM against BB. ELISA assays were performed according to the manufactures’ instructions. For IFA antigen w a s used s tr a i n of B. aftelii, the most fiequent Slovenian isolate. Results: The sera of group of healthy persons were IgM 97.2% negative by IFA, 82.7% negative by Behring ELISA and 90.6% by Dako ELISA. IgG were 87% negative by IFA, 56.3% negative by Behring ELISA and 84.9% negative by Dako ELISA. The sera of patients with erythema migrans were IgM 80% negative by IFA, 37.5% negative by Behring ELISA and 52.5% by Dako ELISA. IgG were in this group 80% negative by IFA, 57.5% negative by Behring ELISA and 65% by Dako ELISA The sera of patients with other manifestations of Lyme disease were IgM negative in 90% by IFA, 55.7%by Behring ELISA and 54.4% by Dako ELISA. IgG were 64.5% negative by IFA, 38.6% by Behring ELSA and 50% by Dako ELISA. Conclusions: The best sensitivity and specificity have been achieved by home-made IFA. The commercial ELISA tests should be used for serological screening because of its increased sensitivity.
V. Maraspin, E Strle, S. Lotrit-Furlan, J. Cimperman, E. Rukit-Sabljit. University Medical Centre, Ljubljana, Slovenia Objectives: To assess the course and the outcome of antibiotically treated immunocompromised patients (ICP) with erythema migrans (EM).
Methods: In the period b m 1991to 1995,49 adult patients with typical erythema migrans and previously identified immunocompromised condition were investigated and followed-up for one year at the Lyme borreliosis Outpatients’ Clinic, University Department of Infectious Diseases, Ljubljana, Slovenia. Results: There were 26 females and 23 males, aged from 17 to 77 years (medan 58 years), suffering from dfferent causes of immunodeficiency. The results were compared with 49 previously healthy age, sex and antibiotic therapy matched persons with an EM. Clinical characteristics of Lyme borreliosis (LB) before treatment were similar in both groups. The majority of patients were treated with an oral antibiotic, only three (6.1%) ICP and two (4.1%) controls received an i.v. antibiotic because of signs of early dissemination of LB. The median duration of EM after the institution of therapy was 10 days (2-90) in the ICP group and 14 d a y s (2-90) in the control group (nonsigdcant difference). Among the ICP five (10.2%) patients developed minor and three (6.1%) major manifestations of LB, while in the control group in three patients (6.1%) minor manifestations were observed and none developed major d e s t a t i o n s ; all required a retreatment with an i.v. antibiotic. Conclusions: The course and the outcome of early LB in immunocompromised patients seem to be sirmlar as in normal population.
[p605] CefuroximeAxetil (CEF), Doxycycline (DOX) and Amoxicillin (AM) for Treatment of Solitary Erythema Migrans (EM) E Strle, V. Maraspin, S. Lotrit-Furlan, E. Rukit-Sabljit, T. Jurca, J. Cimperman. University Medical Centre, Ljubljana, Slovenia Objectives: To compare effectiveness and adverse effects of cefuroxime axed, doxycydine and amoxicillin for weatment of EM. Methods: The study was prospective and began in 1994. For the presentation in this report 210 adult patients with previously untreated typical solitary EM were selected, i.e., the first 70 patients treated with CEF (500 mg bid for 15 d a y s ) , DOX (100 mg bid for 15 days), or AM (1000 mg tid for 5 days, followed by 500 mg tid for additional 10 days) who were followed-up for one year. Results: The median duration of s h lesions after the institution of treatment was 10 (3-21) days in the CEF group, 11 (3-30) days in the DOX group, and 9 (2-28) days in patients treated with AM (nonsigdcant diflerences). During the follow up of 12 months 2 patients (1 in DOX and 1 in AM group) developed major later manifestations of Lyme borrehosis, and in 42 (20%) patients minor manifestations appeared in 13 (18.6%) treated with CEF, in 17 (24.3%) receiving DOX, and in 12 (17.1%) given AM. Bomlia burgdorjm’ sensu lato w a s isolated from the periphery of EM lesions in 23/62, 30/65, and 33/68 patients before treatment with CEF, DOX, and AM, respectively. In 1/30 culture positive patient in DOX group, in 1/23 in CEF group and in 0/33 in AM group B. burgdorfoi sensu lato was isolated again b m normal appearing skin at the site of previous EM 2 months after institution of antibiotic therapy. Seven, 11, and 6 patients receiving CEF, D O X and AM, respectively, reported mild to moderate gastrointestinal discomfort. In addition, 8 patients ti-eated with DOX developed photosensitivity.
Lyme borreliosis
Conclusions: CEF, D O X and AM have similar effectiveness, but adverse effectsare sigtllficantlymore fkquent in patients treated with DOX.
I P606 I Treatment Failure in Erythema Migrans B. Sibanc, G. LegniLar, S. Cvitan,J. Blatnik. Department oflnfectious Diseases, General Hospital, Cefie, Slovenia
Objectives: We talk about erythema migrans (EM) treatment failure whenever we see: 1. EM persistence 2. EM reoccurrence, 3. persistence or development of extracutaneus sequelae (major signs and minor symptoms), 4. persistent increase of antibody titers, 5. persistence of Borrelia burgdoferi. Methods: EM has been treated in General hospital Celje - Infectious department since 1981. In years 1994-1996 544 lyme patients were treated. 323 of them had typical EM and consequent progress in lyme cksease, whlch inckcate the need for hospital treatment with ceftriakson (Lendacin). Among them, we found 51 properly treated EM patients, who show progress of illness. 28 patients were treated with azithromycin, 7 with doksicyclme, 6 with penicilin V, 4 with consequent administration of two proper antibiotics (ATB), 6 with other ATB-s. Results: Only 10 patients had minor signs at the time ofEM persistence and 13 were seronegative at the end of our study. 6 patients (5 children) developed disseminated EM, 11 arthritis, 3 meningoencephaiitis, 1 pericarditis, 1 facial palsy, 29 minor symptoms which persisted more than 3 months or even upgraded. Conclusions: AU patients were treated with cefniakson 2 g/d i.v. 2-4 weeks. After that time, 25 patients were cured, 20 patients improved partially but in 6 patients there were no signs of improvement
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OspC wxs identified more often than OspA. In the late stage antibody response against OspA was more fiequent than against OspC. Among cross-reacted sera the greatest part of B. burgdoferi antigens were discerned h m syphilitic sera.
Marseilles Fever and Lyme Borreliosis L. Angelov, T. Rakadjieva, R. Komitova, M. Pishmisheva, I. Hristova. Higher Medical Institute, Plovdiv, Bulgaria Some of our recent epidemiological studies have shown that human cases of Lyme borreliosis (LB) are reported h m areas where the environmental conditions are not optimal for the life cycle of the tick species I. ricinus. These areas are recognised as foci of Marsedles fever. It is known that the symptoms of LB vary h m patient to patient and often mimic other conditions. For this reason it is &cult for the clinician to distinguish between the two infectious disease. Objective of the study: To analyze the clinical manifestations of LB and Marseilles fever in a random sample of patients. An effort is made to establish the best therapeutic approach to these patients. Methods: The techniques of indirect immunofluorescence and ELISA. Results: 22 patients living in an area recognised as endemic for Marseilles fever were studied. Some of them developed clinical symptoms of LB and were serologically positive for infection with B. burgdoferi. Some domestic animals also tested positive for LB and Marseilles fever. Conclusions: The epidemiological evidence presented in this study gives us the grounds to consider that coinfections with B. burgdo@ and R. conori are possible. A new approach to the treatment of such patients is outlined.
at all.
Disappearance of EM after treatment does not necessarily mean cure of Lyme borreliosis, even when we use "proper ATB-s" long enough
I P609 I Clinical Manifestationsof Lyme Disease in
I P607 I Electrophoreticand Immunologic
N. Trofimov V Scherba L. Titov , E. Danilov A.I. Korzan *. 'Byelomssian Research Institute for Epidemiology and M i ~ o b i o l o ~ , Minsk, Republic of Belarus, 'Clinical Hospital of Brest Region, Republic of Belarus
Belams
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Characterizationof6orrelia burgdohn Isolated in Bulgaria I. Christova, M. Polianova, L. Froloshka. National Center oflnfecfious Diseases, Sofa, Bulgaria Objectives: To basically characterize Bulgarian B. burgdoferi isolates as well as humoral immune response in Lyme dIsease patients against them. Methods: Thirteen Bulgarian B. burgdorferi isolates /11 h m I. rioinus ticks and 2 h m skin biopsies from Erythema migrans patients/ were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis /SDS-PAGE/ and by immunoblotting with sera h m patients with Lyme disease. Cross-reacted sera from patients with syphilis, leptospimsis and lupus erythematodes served to discriminate between specific and nonspecific bands in immunoblots. Results: Regarding Merent species-specific B. burgdoferi OspA/OspB electrophoretic migration, basic identification of the isolates was made. Early humoral immune response in Lyme disease patients was found to be directed mainly against OspC and 41 kDa antigen, and very rarely against OspA. In the late stage of the disease antibohes were proven especially against 41 kDa, 66 kDa, OspA and only in single cases against OspC. Conclusions: SDS-PAGE characterization of Bulgarian B. burgdorjki isolates revealed considerable predomination of B. garinii both among tick and skm isolates. By sera h m patients with Lyme disease the following major antigens in the isolates were proven 66 kDa, 41 kDa, OspA, OspC. In the early stage of the disease
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Objectives: To study prehmnary data on characteristics and peculiarities of Lyme Disease (LD) course in Byelorussian population. Methods: 24 patients with LD were examined by us in 1995-1996. The disease was diagnosed on the basis of migrating erythema (ME) in anamnesis, revealed during a physician's examination or h m the patients complaints. It was verlfied by detecaon of antibodies against Borrelia burgdofm' in blood sera with the help of indirect immunofluorescence assay Results: ME was a most fiequently occurred symptom h m all clinical d e s t a t i o n s (87%). Cutaneous manifestations were accompanied by common infectious syndrome in 10 cases (47%), that was expressed in fever of different rates, as well as in head, muscle and joint pains. Disturbances in cardIo-vascular system were reported in 9 patients (38%), they were attended by unpleasant sensation and pains in heart. Various rates of blockade in conducting system and atrial extrasystoleswere also identified in elecaocardiogrammes.The signs of anicteric hepatitis in the form of nausea, feeling of heaviness, located in right hypochondrium, enlargement of liver size and elevation of transaminase levels 2-3 times were established in 3 patients (12%).Serous meningitis ofborrelia etiology was diagnosed in 4 patients (17%). Conclusions: ME and common infectious syndrome are main clinical signs of LD. Lower prevelance of other symptoms, which are typical at late stages, appears to be conditioned by the early administration of antibiotics to patients.
Journal of Clinical Microbiology and Infection, Volume 3 Supplement 2
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1 P610 I Prevalence of Bomlia burgdohti Antibodies in Urban and High Risk Areas
D. Tuncer',D. Colak', E Saym2,D. O@inc', C. Ergin', B. Tuncer3, G. Mutlu'. 'Akdeniz University CIin. Microbiology D q t , Antalya, Turk% 'Ankara University Veterinary Faculty, Ankara, Turkey, 3~tare Hospital Finike, Turkey Objectives: An epidemiological study on Lyme disease was made by comparing the populations of mountain villages and city centers. Methods: 244 subjects h m mountainous areas and 124 subjects finm city centers were examined with respect to Lyme &ease. B. burgdofm' IgG antibodies were searched for in the sera of subjects by microelisa technique. Positive results were later confirmed by Western blot techmque. Ticks we^ collected h m goats and sheep, which are in close contact with the people in endemic regions, and presence of Ixodes species, which are the reservoir of B. burgdorfen' was inveswted. Results: By microelm, 22.1% of rural and 6.4% of urban populations were shown to have B. burgdorferi IgG antibodies. By Western blot 80.6%of positive cases confirmed to be positive. 80.4% of ticks were idenhfied as Ixodec ricinus Conclusion: This study suggests that Lyme disease might be considered as endemic in rural areas of Antalya.
I P611 I Standardization of Western Blotting in Lvme Borreliosis (LB) A. Lakos. Lyme Disease Center, Budapest, Hungary Objective: New standardization of Western bloning in LB, specification of specific and aspecific bands, using Borreha ahelii (BA) as antigen. Methods: The proteins of BA (ACA1) were separated by PAGE and blotted onto Immobilon membrane. The immune reaction was carried,outwith 100 serum samples each of the following clinically defined groups: erythema migrans - EM (l),Bannwarth-syndrome - BS (2), acrodermatitis chi. atrophicans - ACA (3), infants (4), healthy blood donors - HBD (5), forestry workers (6) and uveitis patients (7). Groups 1-3 represent Merent clinical manifestations of Lyme &ease, groups 4-7 served as control. IgM and IgG antibodies were tested separately. In group 2, CSF samples were also evaluated. Altogether 1600 tests were done. The intensitiy of bands was estimated and scored finom 0 to 3. Location of bands was defined with monoclonal antibodies. Statistical evaluation was done by EPINFO and SPSS PC. Results: IgM antibodies against both the 22 kD and 41 kD protein was found to be 100% spenfic. However, IgM testing has a low diagnostic value, because only five EM and nine BS patients had IgM but no IgG antibodies. Anti-flagellar (41 kD) and anti-39 kD IgG antibodies were not spec& at all, in contrast with many previous studies. Specfic bands were 22; 29; 35; 44; 47; 49; and 93 kD. The best specificity/sensitivity ratio was found when cut-off level was s e t up at score 1.5 (one strong plus one faint reaction at the above-mentioned bands). According to t h criteria, ~ ~ only three HBDs and no d a n t s were positive. The sensitivity of IgG tests among consecutive EM and BS patients was 40 and 63%. respectively. In BS, when both CSF and serum, IgG and IgM were evaluated, the sensitivity was 88%.After six weeks the first symptoms, all BS patients were positive as well Y the ACA patients. Conclusion: We found a specificity pattern difierent h m previous studies. Our results suggest that specific Western blot pattern may vary strain by s m i n and there is no universally valid specific Western blot bands. This work should be repeated for any borrelia isolate candidate for dugnostic purpose.
(p612(Ti-Borne Borreliosisor Infectious
Mononucleosis- A Diagnostic Challenge in Children
L. Krbkwi M. KapZkovi *, I. Stefflovi', J. Kunicki 9.G. Mendel Children's Hospital, Univmity of Bmo, Czech Republic, 'Hygiene Institute, Bmo, Czech Republic Objectives: To examine IgM seropositivity against Bonelia burgdorfm' (Bb) with nonspecific symptoms for tick-borne borreliosis in
pediatric patients. Methods: 217 children were seen h m 1994 to 1996 for IgM seropositivity against Bb. Erythema migram, borrelial lymphocyt o m , neuroborreliosis and Lyme arthritis were diagnosed in 165 children. We analysed 35 children for possibly cross-reacting antibodies to rheumatoid and antinuclear factor, to viral capsid and nuclear antigen for Epstein-Barr infection. Enzyme-linked immunosorbent assays were used to dqnose Bb or EB infection. Westernblots or IgM antibody capture tests are not used routinely in primary care practice. Results: 26 girls and 9 boys, ages 3 to 18 years with cervical lymphadenopathy, fever, fatigue, cephalea or tonsillitis were repeatedly examined. The atypical lymphocytes were found in 16/35 (46%)patients. 10/35 (29%) children showed elevated transaminases.20/33 (61%) children had positive IgM antibody to viral capsid antigen for infectious mononucleosis in the I. sample and only 6/35 (17%) had it in 11. During the convalescent phase 54%-83% children developed I g G antibodies to EB nuclear antigen. Antinuclear and rheumatoid factor were negative in all patients. Conclusions: Tick-borne borreliosis and EB infection may both be associated with lymphadenopathy or other common features of illness. Cases with nonspecific symptoms, IgM seropositivity to Bb and those with tick bite history lead to erroneous diagnosis of tick-borne borreliosis. IgM seropositivity to Bb in 35/217 (16%) children with infectious mononucleosis supports the knowledge about false-positive serology in diseases with polydonal B cell activation.
(p613j Evolution of the Immune Responsein M i e n t s with Erythema Migrans G. Grabher, B. Redl, P.Loidl, G. Sto5er. Instifute o f M i m b i o l o ~ , Uniwrsity oflnnsbruck, Medical S h l , Austria Objectives: To assess the evolution of the immune-responses of patients with EM during the course of disease using immunoblots (IB) with extracts from three Borrelia genotypes and recombinant proteins. Methods: A total of 259 serially collected sera b m 97 phyucian documented patients with known duration of EM were analyzed by immunoblotcing for IgM and IgG, using extracts b m strains B31 (B. burgdofm' sensu stricto), PKo (B. afzeliil), PBi (B. garinit), recombinant proteins OspA, OspB, OspC, flagellin (and two Merent central hgments P41i), P39 and P100/83 h o r n B. garinii and a commercial DotBlot. Evaluation was, if applicable, as recommended by CDC and ASTPHLD. Results: From 45 patients which presented with EM of less than 7 days in duration 8 (18%) were IgM positive in IB,whereas h r n 52 patients with EM of 7-14 days in duration 47 (90%) were IgM positive. In total, the serum samples b m 88 (91%) of the patients revealed a seroconversion within 2-4 weeks after appearance of EM. IgM to OspC and flagellin remained detectable for long periods, up to 6 month in some patients. Conclusions: The appearance of IgM in Il3 correlated directly with duration of EM. The Merences in the reactivities with OspC
Lyme borreliosis
in dependence of the genospecies used for IB were generally compensated by detection of antibodies to P39, P41i and/or P100/83.
I P6141
Urinary Excretion of 6.burgdorfefiDNA in Patients with Lyme Borreliosis
B.L. Schmidt, E Breier, E. Aberer. L B I f dermato-wenerolog, Serodiagnosis, Vienna, Ausfria Objectives: To determine the urinary excretion of B. burgdoferi (Bb)-DNA in patients with Lyme borreliosis after therapy. Methods: Urine samples (n = 442) h m 134 patients (pat.) with uncomplicated erythema migrans (EM) or with EM accompanied by systemic symptoms, fever, chills, paresthesia, arthralgia or myalgia @MA), were tested before and up to one year after therapy by a nested PCR for the presence of Bb-specific DNA. Results: Immediately after end of therapy, 79% of pat. were positive by PCR. After 6 months, all but 9 were non reactive. Reevaluation of these 9 pat. showed interruption of treatment or treatment duration of only 5-10 days in 7 pat.. All could be retreated for 20 days with ceftriaxon and urine controls after three months were negative by PCR. Conclusion: Sensitive PCR-tests can demonstrate Bb-DNA in urine of pat. after matment. A persistent reactivity six months later indicates treatment failures.
I P615 I
Systemic Reaction in Patients Treated with Cefotaxime for Lyme Disease
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I P616 I Detection of 60rrelia burgdohriDNA in Muscle of Patients with Chronic Myalgias Related to Lyme Disease
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M. Frey I.', J. Sibha 3 , Y. PiCmont L. Marcellin ', E? Boohs , E? Vautravers M. Jesel', J.L. Kuntz H. Montes', B. Jaulhac '.
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'Services de Mbdecine Physique et Rhadaptation, Strarboutg, France, 'd'Explorations Fom'onnelles Neuromusculaires, Strarbourg, France, 'de Rhumatologie, Strasbourg, France, 4d'Anatomo-Pathologie Ghnhafe, Strarboutg, France, 'Institut de Bactk.iologie, Strarbourg, France
Objectives: Chronic myalgias related to Lyme disease are generally considered as sequelae. Our objectives were to establish whether Bonelia burgdorferi (E?. burgdoferi) may persist in the muscle of patients in such a case and to evaluate the contribution of a PCR assay for detecting this bacteria. Methods: In a prospective case-control study, B . burgdoferi DNA detection was performed by PCR on muscle biopsies of 8 consecutive patients with chronic widespread myalgias related to Lyme disease. The PCR assay used a specific chromosomal fragment of the flagellin gene. Other classical causes of myalgias were ruled out. Muscle samples from 14 controls were also tested. Results: B . burgdo& DNA was detected by PCR in muscle fium 4 of the 8 patients but not in controls. Three of the 4 positive patients had previously received an antibiotic treatment for Lyme disease; PCR analysis was done 3 months after antibiotic treatment for 2 of these 3 patients and 38 months for the thud one. Conclusion: Chronic myalgm related to Lyme disease can be associated with persistence of B. burgdofm' DNA in muscle, even despite previous supposedly curative antibiotic treatment.
Ch. Jvlichel J.-E? Collet2. ' D u g Surveillance Dept., Hoe& AG, Frankfurt, Germany, 'Dept. of Epidemiology and Biostatistirs McGill University, Montreal, Canada
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Objectives: Starting in 1989 a couple of adverse reaction reports were received fium the US in which the occurrence of a new systemic reaction - also reported as "delayed Jarisch-Herxheimer Reaction" - was idenhfied in patients treated with cefotaxime for Lyme disease. This study was undertaken to investigate whether this reported systemic reaction could be considered as a new adverse drug reaction to cefotaxime or the expression to the underlying Lyme disease. Methods: All spontaneous adverse reactions reports received by Hoechst AG h m the US within the period 01/Jan/87 to 31/Dec/93 were reviewed. An operational definition of the observed systemic reaction was established and reports were classified in five categories according to the probabihty to represent a case of the reaction: certain, possible, unlikely, highly unlikely or excluded. Results: A strong association between Lyme &ease as the indication for cefotaxime treatment and the systemic reaction was detected 97% (30/31) of the cases identified as certain were treated for Lyme disease, as opposed to 90% (27/30), 69% (31/45), 39% (651167) and 11% (42/392) of those idenhfied, respectively, as possible, unlikely, highly unlikely and excluded. Conclusions: This suggests that the new reported systemic adverse event may not be due to cefotaxime on its own but that it may either represent a combined effect of the drug and disease or persistent symptoms of the underlying Lyme disease not resolved by antibiotic theram. The possibility of an effect of long treatment duration of cefotaxime cannot be completely ruled out, but remains very unlikely.
I. Nahimana , 0. Peter *, L. Gern G. Praz F? Francioli . 'CHVY Lausanne, Switzerland, 'Insf. Central des Hbpitaur bblaisans, Sion, Switzerland, Unioersid de NeucMtel, Switzerland
Risk of Lyme Bomliosis after a Tick Bite in the French Part of Switzerland
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Objectives: To evaluate the risk of acquiring Lyme borreliosis after a tick bite and to correlate the risk with the presence of Borrelia sp
in the ticks. Method Since 1993, physicians of the French part of Switzerland were asked to report on all patients consulting for a tick bite. The protocol included a standardized questionnaire, two blood samples collected two months apart and mailing of the tick if available. Serology was performed with both a commercial test (Lyme IgG + M, Vidas Biomerieux) and a westernblot assay detecting both IgG and IgM. Criteria for positive immunoblot were a minimum of 5 bands including flagelhe and 2 of the following specific bands: P39, OspA, OspC 93 kDa. Seroconvenionwas defined by passing h m a negative immunoblot result to a positive one at the second sampling. Ticks were analyzed either by immunofluorescence or PCR with species-specific primers. Results: Among 510 patients who had a negative serology at the first visit, 40 (8%) seroconverted. The rate of seroconversion was 4% in the 297 patients who consulted because of the tick bite only (group I), 7.5% in the 121 patients with a s m a l l local reaction at the site ofthe bite (group 2) and 21% in the 92 patients who already had a lesion qu-g (25 cm) for an erythema migrans (EM): (group 3). Among the 418 patients of group 1 and 2, 8 (2%) developed an EM, among which 4 seroconverted. Among 179 analyzed ticks, 26 were positive. No correlation could be found between Bonelia sp in ticks, and clinical and/or serological results. Conclusions: In patients consulting for a tick bite without EM (group 1 and 2), the rates of seroconversion and EM were 5% and 2%, respectively. Seroconvenion and subsequent EM was more ke-
Journal of Clinical Microbiology and Infection, Volume 3 Supplement 2
146
quent in patients with small local lesions (group 2). suggesting that these lesions often represent Bonefia sp infection at an early stage.
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Conclusion: The protein recognized by D6-Mab is Uely an outer surface protein (OSP) so far undescribed and we are in the process of characterizing it, knowing its imporrance for species typing and dagnosis.
[P618 Association of Distinct Clinical Manifestationsin Chronic Lyme Bomliosis with Bonelia burgdorhri sensu stricto, B. garinii and B. afzelii as Evaluated by lmmunoblot
0. Peter, A.G. Brea, E. Dayer. Institut Central des HGpitaux Ihlaisans, Sion, Switzerland
Objective: To evaluate the serologic reactivity ofpatients with Lyme arthritis, neuroborreliosis and Acrodermatitis chronica atrophicans (ACA) by immunoblots using B. burgdofm' sensu strict0 (ss), B. garinii and B. afrelii as antigens. Methods These 3 borrelia species are transmitted by Ixodes ricinus in Europe. A total of 81 patients with chronic symptoms of Lyme borreliosis (28 patients with arthritis, 28 with neuroborreliosis, 25 with ACA) were analysed by immunoblots using B. burgdofen ss, B . garinii and B. afzelii as antigens. In order to give a semi-quantitative appreciation of the reactivity, we scored (0 to 3 points) the intensity of the reaction to seven borrelia antigens ranging from 18 kDa to 93 kDa. Results: The serologic reactivity of patients with chronic manifestations of Lyme borreliosis was as follow: 62% of patients with neuroborreliosis reacted more intensively with B. garinii, 71% of patients with arthritis reacted with B . burgdogm' ss and 80% of patients with ACA reacted with B. afzelii. Usually criteria for positive immunoblot were reached whatever the Borreha species was. The stronger reactivity for one species was visually obvious and scores helped to objectivate the readings. Conclusions: These associations between the symptoms and the preferential serologic reactions to Werent Borrelia species may give additional arguments for the diagnosis of chronic Lyme borreliosis.
I P619 I Characterization of a Low Molecular Weight Lipoprotein Specific of B. garinii, Recognized by the D6-Mab E. Dayer, A.G. Brea, 0. Peter. Imtitut Central des HGpitaux Valaisans, Sion, Switzerland
Objective: To characterize the epitope recognized by D6-Mab specific of B. garinii. Introduction and Methods: Specific symptoms of chronic Borrekosis have been related to persistent infection with Herent Borrelia species. In Europe, neuroborreliosis has often been associated with B . garinii infections, while other symptoms, acrodermatitis chronica amphians (ACA) and arthritis, have been associated with B. afrelii and B. burgdofm', respectively. Typing the Werent isolates by their reactivity to spec& monoclonal antibodies is possible by immunoblotting. We used standard SDS-PAGE 1D and 2D gels with PVDF membrane transfer, as well as N-terminal sequencing to characterize protein. Results: D6-Mab recognized specifically B. garinii; the specific antigen is a surface lipoprotein of 11.2 kD and an estimated pkl of 5.7. Its N-terminal portion sequenced h m PVDF transfer membrane gave the following sequence: MVKKEAIIKAVNGLLVXP: The search for homologies revealed no identity and homologies of only 40% with POL-SOW3 non structural protein and with other ribosomal and peripksmic proteins. Using polyclonal antisera from chronically infected patients, we were able to show a major reactive protein in the same kD range in other Borrelia species, although it migrates in slightly Nerent position according to species.
1 P620 1 Developmentand Evaluationof Confirmatory Tests for Lyme Disease Using an Extract of B. garinii
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K. Gimrdet L. Binet', 0.Peter'. 'Imtifut Central des Hdpitaux Vafaisans,Sion, Switzerland, 'Lubodia S.A., Em, Switzerland Lyme hsease serology remains extcemely complex and positive resdts by ELISA require confirmatory tests (immunoblot or PCR). Most commercial confirmatory tests currently available are h m US origin and use as target antigens e m c t s from Borrelia burgdorferi. In Western Europe, s t r a i n prevalence is Herent and w e have developed two immunoblots (IgG and IgM) with an extract of B . garinii. These kin allow the identification of antibodies against proteins of 93, 60, 41, 39, 37,32.5,22-23 and 18 kD. C h c a l tr& organized in Switzerland have shown large geographcal variations in the seropositivity rate of the overall population. The speciticity of the luts has been evaluated for possible cross-reactions with syphilis or EBV antibodies and with autoantibodies. The sensitivity of the IgG immunoblot reaches 100% for Lyme arthritis, 96% for acrodermatitis, 90% for erythema migrans and 89% for neuroborreliosis.
Toxoplasma gondii
I P621 I
Prevalence of Toxoplasma Gondii Antibodies in Young Females of La Plata, Argentina
J. C o r d h i , M. Rub, G. Alberich, J. Contarelli, L. Massera, M. Michaan, M. Garcia, S. Cabrera. Infecous Diseases Department, Hospital San Juan de Dios, La Plata,Argentina Objective: To determine the seroprevalence of Toxoplasma gondii (TG) antibodies in urban and suburban young females finom La Plata, Argentina. Subjects and Methods: Samples from 271 consecutive healthy females were collected between February to October 1995. Serum was tested with indirect Hemaglutination with and without 2-mercaptoethanol. Positive test were confirmed with enzyme linked immunoassay. Results: The meda age was 22 (range 15-45). 156 (57.56%)living in the city, and 115 (42.43%) in a rural area close to La Plata. No Werences were found for social, cultural, religious or economical conditions between the groups. Dietary habits and frequency of contact with atmnals (including pets) were Werent (p 0.005). A global seroprevalence of 43.5% (118/271) was found. Values were higher in the rural group: 53.5% than in the urban (36.8%) p > 0.009. Moreover, the prevalence increase with the age, with significative a e r e n c e up to 33 yrs. (p = 0.008). Commentary: Suburban residence enhance the possibility to get TG as simJar as dietary habits and close contact with animals. The seroprevalence raise with the age. Because 56.5% of females were noreactives at wedding time, screening programs are necessary for pre-nuptial detection.